The present invention relates to the production of covalently closed circular (ccc) recombinant DNA molecules. Such molecules are useful in biotechnology, transgenic organisms, gene therapy, therapeutic vaccination, agriculture and DNA vaccines.
With the invention in mind, a search of the prior art was conducted. E. coli plasmids have long been the single most important source of recombinant DNA molecules used by researchers and by industry. Today, plasmid DNA is becoming increasingly important as the next generation of biotechnology products (gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. Plasmid DNA vaccines may find application as preventive vaccines for viral, bacterial, or parasitic diseases; immunizing agents for the preparation of hyper immune globulin products; therapeutic vaccines for infectious diseases; or as cancer vaccines.
The basic methods for obtaining plasmids (by bacterial fermentation), and for their purification by the alkaline lysis method are well-known (Birnboim, H C, Doly J. 1979 Nucleic Acids Res. 7: 1513-1523). Initially, the fermented bacterial cell paste is resuspended and lysed (using a combination of sodium hydroxide and sodium dodecylsulfate), after which the solution is neutralized by the addition of acidic salt (e.g., potassium acetate), which precipitates the bacterial DNA and the majority of cell debris. The bulk of super-coiled plasmid DNA remains in solution, along with contaminating bacterial RNA, DNA and proteins, as well as E. coli endotoxin (lipopolysaccharide, or LPS).
Alternatively lysis using heat/lysozyme treatment in the presence of nonionic detergent has been used to release intact plasmid DNA. Cells can also be lysed, and nucleic acids released, using high pressure exposure to supercritical fluids or by treatment with organic solvents, or detergents.
These lysis methods release cell impurities, which then require purification steps to remove. As well, the alkaline lysis or heat denaturation cell lysis methodologies currently utilized in plasmid DNA manufacture are costly, inefficient, and create large toxic waste-streams. Alternative, cost effective lysis methods have not been developed.
The soluble fraction is then separated by filtration and subjected to a variety of purification steps, which may include: RNase digestion; chromatography (ion exchange gel filtration, hydroxyapatite, gel filtration, hydrophobic interaction, reverse phase, HPLC, etc.); diafiltration; organic extraction, selective precipitation, etc.
Exemplary downstream plasmid purification processes after lysis described in the art reduce genomic DNA levels to 0.01-1% or less. The following processes described in the art are not an exhaustive list, and include the specified genomic DNA reduction steps: 0.01% genomic with hydroxyapatite (Wils P, and Ollivier, M. 2004 U.S. Pat. No. 6,730,781), 0.05% genomic with hydrophobic interaction chromatography (Nochumson S, Durland R, Yu-Speight A, Welp J, Wu K, and Hayes R. 2001 US Patent Application 2001/0034435; Diogo M M, Querioz J A, Monteiro, G A, Martins S A M, Ferreira, G N M, and Prazeres D M F. 2000 Biotech Bioeng 68:576-583), 1% genomic with ammonium sulfate precipitation (McNeilly D S. 2001 U.S. Pat. No. 6,214,586), 0.2% genomic with size exclusion chromatography (Lemmens R, Olsson U, Nyhammar T, and Stadler J. 2003. J Chromatography B 784:291-300), <1% genomic with Tangential flow ultrafiltration (Bussey L B, Adamson R, and Atchley A. 2000 U.S. Pat. No. 6,011,148), <1% genomic with differential polyethylene glycol precipitation (Marquet M, Horn N, Meek J, and Budahazi G. 1996 U.S. Pat. No. 5,591,064), CTAB precipitation and gryolite LRA absorption (Lander R J, Winters M A, and Meacle F J. 2002 US Patent Application 2002/0151048), 0.1% genomic with triple helix chromatography (Crouzet J, Scherman D, and Wils P. 2001 U.S. Pat. No. 6,287,762).
The introduction of plasmid DNA into humans presents some special considerations and challenges, which have been addressed in FDA regulatory guidance, including Points to consider on plasmid DNA vaccines for preventive infectious disease indications (Food and Drug Administration, Center for Biologics Evaluation and Research. 1996 Points to consider on plasmid DNA vaccines for preventive infectious disease indications DOCKET NO. 96N-0400, and Food and Drug Administration, Center for Biologics Evaluation and Research. 1998 Guidance for industry: Guidance for human somatic cell therapy and gene therapy.). These documents indicate concerns about the various contaminating substances, and suggest tests that can be used to assess the levels of each contaminant. The guidance documents stop short, however, of suggesting maximum acceptable levels of contaminating RNA, DNA or proteins, as these are not known. However, the allowable limit for genomic DNA would be 0.00001% if the 100 pg genomic DNA/dose specification currently required by FDA guidelines for recombinant protein drugs (FDA. 1993 Points to consider in the characterization of cell lines used to produce biologics) were applied to a 1 mg DNA vaccine dose. This is several logs lower levels than standard large scale plasmid DNA preparations (0.01-5% genomic DNA) and cannot be attained using currently available cost effective manufacturing methodologies. New methods are needed to afford further reductions in genomic DNA.
Nucleic acids can be eliminated early in the process (e.g., by nuclease digestion), or later (e.g., by chromatographic separation). A relatively common practice, until recently, was the use of bovine pancreatic ribonuclease (RNase A) in the lysis buffer, to degrade RNA. Although it was reasonably effective in reducing the quantity and size of RNA, it also introduced the bovine-source RNase, which is undesirable from a regulatory standpoint, as it could be contaminated with prion agents, notably with the bovine spongiform encephalitis (BSE) agent. Indeed, there is a growing desire to perform fermentations and purifications of bacterial products (intended for human or animal use) entirely under animal product free (APF) conditions.
Presently, we know of no highly effective commercial enzymes for specifically degrading E. coli genomic DNA while leaving super-coiled plasmid intact (‘plasmid-safe’ nuclease). Occasionally, however, nucleases, such as the ATP-dependent Rec BCD exonuclease enzymes (Qiagen Large Construct Kit Handbook, June 2003; Wahle, S, Schorr J, and Weber M. 2001 U.S. Pat. No. 6,242,220; Isfort R J 1992 BioTechniques 12: 798-804) are added to partially purified plasmid DNA preparations. In a related approach, the crude plasmid preparation is heat treated to denature all non-circular DNA to single stranded form, then single stranded exonucleases such as SI nuclease, mung bean nuclease, P1 nuclease, T7 exonuclease, Bal31 nuclease, Exonuclease I, Exonuclease III, Exonuclease VII or Lambda Exonuclease (Hyman E D. 1992 World Patent Application 92/13963) is added. These DNase enzymes cannot be added directly to the lysis (as with RNase), because these enzymes are generally more fragile than RNase, and would be inactivated in an alkaline/SDS environment. Such approaches are therefore expensive and impractical for commercial scale plasmid manufacturing.
In order to overcome the obstacles that exist with adding purified nucleases to plasmid DNA preparations, alternative approaches have been developed that utilize endogenous nucleases to remove genomic DNA. Early methods induced general DNA damage (e.g. ultraviolet radiation in repair deficient hosts (Sancar A, Hack A M, and Rupp W D. 1979 J Bacteriol. 137: 692-693), or ionizing irradiation (MacPhee D G, Radford, A J, and Reanney D C. 1988 U.S. Pat. No. 4,755,464) in which plasmids survive due to a lower probability of damage (i.e. smaller target than the genome) relative to the chromosome; degradation, mediated by endogenous nucleases (e.g. RecBCD), proceeds from the DNA breakage sites in the genome. A more specific system that utilizes restriction endonucleases to cleave genomic DNA has been reported, wherein restriction endonuclease activity is controlled by a thermosensitive methylase. Shifting to the restrictive temperature inactivates the methylase, leading to cleavage of genomic DNA, and subsequent endogenous exonuclease digestion (Hanak, J, Alexis J, and Ward J M. 2001 World Patent Application WO 01/29209). However, the level of genomic reduction is modest with these methods, and plasmids would need to be engineered to lack the relevant restriction sites so this method does not have general utility.
Specialized E. coli strains have been developed, which express recombinant nucleases in the periplasmic space in order not to disrupt E. coli gene expression during cell growth. In one case bovine pancreatic RNase is directed to the periplasmic space by means of a secretion signal, Upon lysis, the RNase becomes mixed with the RNA, degrading it (Cooke G D, Cranenburgh R M, Hanak J A J, Dunnill P, Thatcher D R, Ward J M. 2001 A J. Biotechnology 85: 297-304). This system is utilized to reduce RNA levels during alkaline lysis. No reduction in genomic DNA is afforded by this method. Similar systems to overexpress periplasmic Staphylococcal nuclease (Cooke G D, Cranenburgh R M, Hanak J A J, Ward J M. 2003 J. Biotechnology 101: 229-239; Huisman G W, Luo L Z, and Peoples O P. 2004 US Patent Application 2004/0014197; Boynton Z L, Koon J L, Brennan E M, Clouart J D, Horowitz D M, Gerngross T U, and Huisman G W. 1999 Pseudomonas putida. Appl. Environ. Microbiol. 65:1524-1529), or endogenous E. coli EndA periplasmic nuclease (Leung W S, and Swartz J R. 2001 U.S. Pat. No. 6,258,560) have been developed, to reduce nucleic acid contamination of protein or other biomaterial preparations. These systems are not plasmid-safe, and require gentle protein purification processes and buffers for activity. The induction of plasmid-safe DNases in fermentation culture is discussed in theoretical context by Kelly 2003 (Kelly W J. 2003 Biotechnol Appl Biochem 37:219-223) but a methodology or nuclease is not specified.
Autolytic cell lines have been developed to facilitate protein production (Leung and Swartz, Supra, 2001). In this cell line, lysozyme is expressed by the cell in the cytoplasm, and released to the periplasm at the desired time by co-expression of a holin (membrane spanning peptide or protein) that creates a channel allowing leakage of lysozyme, and other cytoplasmic proteins, from the cytoplasm to the periplasm. Example lysozyme/holin combinations that can be utilized are known in the art. Some lysozyme/holin combinations are discussed in Young 1992 (Young R. 1992 Microbiol. Molec. Reviews, 56: 430-481) and included herein by reference. The phage lambda lysis proteins have been used in autolytic cell lines for the production of proteins (Leung and Swartz, Supra, 2001).
Autolysis conditions, as opposed to alkaline or heat lysis, do not selectively denature genomic DNA. The product of lysis is very viscous, creating processing problems. For protein production, non specific nucleases are added, or expressed periplasmically in the strain (e.g. endA nuclease Leung and Swartz, Supra, 2001; Staphylococcus nuclease; Cooke et al, Supra, 2003, Huisman et al, Supra, 2005, Boynton et al, Supra, 1999) to reduce viscosity after cell lysis. Such systems could not be utilized for plasmid production.
The purification processes utilized in plasmid DNA manufacture are costly, inefficient, and create large toxic waste streams. Residual genomic DNA levels greatly exceed currently acceptable standards for commercial products. These limitations place a cost and purity burden on commercialization of plasmid DNA production processes.
Even in view of the prior art, there remains a need for a cost effective method for genomic DNA reduction. As well, a simplified, less costly purification process which reduces the use of costly or toxic chemicals is needed.